Epidermal growth factor 2 is a transmembrane tumor associated antigen encoded by the HER2/neu proto-oncogene (also called c-erbB-2), which is a member of the epidermal growth factor receptor family of cell surface receptors. The HER2 gene was originally isolated from a rat neuroglioblastoma (Shih et al., Nature 290: 261-264 (1981)) and later cloned and characterized from human cells (Coussens et al., Science 230: 1132-39 (1985); King et al., Science 229: 974-76 (1985)).
HER2/neu is further classified as a member of the HER family of receptor tyrosine kinases, which consists of four receptors that participate in cell growth and differentiation. The HER receptors contribute to maintaining normal cell growth by binding growth factor ligands as dimers. Specifically, human HER2 forms heterodimers with other members of the EGFR family (HER1, HER3 and HER4) (Klapper et al. Adv Cancer Res 77: 25-79 (2000)). Following hHER2 dimerization and tyrosine auto-phosphorylation, docking sites for cytoplasmic signaling molecules are generated and recruitment of second signaling molecules is initiated. Intracellular signaling cascades, which ultimately result in the activation of genes important in cell growth, are thus initiated.
Low levels of expression of the HER2/neu transcript and the encoded 185 kD protein are normally detected in adult epithelial cells of various tissues, including the skin and breast, and tissues of the gastrointestinal, reproductive and urinary tracts (Press et al., Oncogene 5: 953-962 (1990)). Higher levels of HER2/neu expression are also detected in the corresponding fetal tissues during embryonic development (Press et al., supra).
Several observations make the HER2 antigen an attractive target for active specific immunotherapy. First, the HER2/neu gene is commonly overexpressed or amplified in various malignancies, such as carcinomas of the breast, ovary, uterus, colon, and prostate, and adenocarcinomas of the lung (reviewed in Disis and Cheever, Adv. Cancer Research 71: 343-371 (1997)). Overexpression of HER2/neu correlates with a poor prognosis and a higher relapse rate for cancer patients (Slamon et al., Science 244: 707-712 (1989)). Amplification of human HER2 leads to enhanced MAP kinase activity and cell proliferation, and contributes to the aggressive behavior of tumor cells (Ben-Levy et al. Embo J 13(14): 3302-11 (1994)). The high expression level of HER2 observed in tumors is in direct contrast with the low levels associated with normal adult tissues.
Additionally, many cancer patients suffering from malignancies associated with HER2/neu overexpression have had immune responses against the HER2 protein. Anti-hHER2 cytotoxic T lymphocytes (CTL) have been isolated from breast and ovarian cancer patients (Ioannides et al. Cell Immunol 151(1): 225-34 (1993); Peoples et al. Proc Natl Acad Sci USA 92 (14): 6547-51 (1995)). Several HLA-A2.1-associated hHER2 peptides have been defined and peptide-specific T cells can be generated in vitro (Fisk et al. Cancer Res 57(1): 8-93 (1997); Yoshino et al. Cancer Res 54(13): 3387-90 (1994); Lustgarten et al. Hum Immunol 52(2): 109-18 (1997)).
The above findings demonstrate that anti-ErbB-2 immune effector mechanisms are activated in cancer patients and highlight the potential benefit of enhancing such immune reactivity. An effective vaccine exploiting the immune response to HER2/neu must both enhance this immunity to a level that is protective and/or preventive and overcome self-tolerance.
Based on the above recitation, HER2/neu has been pursued as a target for the development of immunological treatments of malignancies. Anti-HER2 monoclonal antibodies have been investigated as therapies for breast cancer, with each antibody approach demonstrating various levels of success (for discussion, see Yarden, Oncology 61 (suppl 2): 1-13 (2001)).
Additionally, DNA and peptide-based vaccines targeting HER2/neu have been reported. Amici et al. (U.S. Pat. No. 6,127,344) disclose a method for inducing immunity against HER2/neu by administering an expression vector comprising the full-length human HER2/neu cDNA functionally linked to the human cytomegalovirus promoter. Morris et al. (WO 2004/041065) disclose a method of vaccination with dendritic cells modified by adenoviral vectors expressing a non-signaling HER2/neu gene. Cheever and Disis disclose methods for immunizing humans against HER2/neu-associated cancers with HER2 peptides (U.S. Pat. No. 5,846,538). Additionally, HER2/neu peptide-based vaccines have been studied in rodent models (for review, see Disis and Cheever, Adv. Cancer Res. 71:343-71 (1997)).
The development and commercialization of many vaccines have been hindered by difficulties associated with obtaining high expression levels of exogenous genes in successfully transformed host organisms. Therefore, despite the identification of the wild-type nucleotide sequences encoding hHER2 protein described above, it would be highly desirable to develop a readily renewable source of human HER2 protein that utilizes hHER2-encoding nucleotide sequences that are optimized for expression in the intended host cell, said source allowing for the development of a cancer vaccine which is efficacious and not hindered by self-tolerance.